The rapid development of molecularly targeted probes for use in vivo has led to growing interest in clinical applications that incorporate information from these probes. In particular, there is a need for techniques to visualize these targeted probes during surgery, particularly to identify tissue either for removal or preservation. When looking at fluorescent probes in tissue, the major difficulty is usually separating the probe fluorescence from the tissue autofluorescence. Because the autofluorescence has different spectral properties than the fluorophore, it can easily be separated using multispectral imaging, in which several full emission spectra can be taken for each pixel. Although the implementation of acousto-optic tunable filters has greatly increased the speed of these techniques, the minimum time required to acquire an image cube is still several seconds with commercially available systems. When imaging using fluorescent probes with lower efficiency or when used at lower doses, the image acquisition time can be as long as several minutes. This data acquisition rate makes the use of multispectral imaging to provide real-time feedback during a surgical procedure impractical. Even when used for diagnostic purposes, the unavoidable motion of the subject during the time required to acquire an image cube can lead to an unacceptable loss of spatial resolution. In years past, this project focused on the development of instrumentation for incorporating fluorescent and multi-spectral imaging into surgical applications, using two major approaches. We developed two prototype instruments. The first prototype enabled real-time visualization of fluorescent probes to guide surgery, evaluated using a murine model of ovarian cancer in which peritoneal metastases were identified using a GSA-Rhodamine G probe developed in NCI. In this instrument, as an alternative to multispectral imaging, aggressive filtering of both emission and excitation light was used to minimize the effects of tissue autofluorescence;the prototype system had comparable sensitivity and specificity to that achieved with a commercial multispectral system, with an image acquisition rate of fifteen frames per second. The second prototype focused on diagnostic applications using multispectral imaging, by providing a white-light stack of images for spatial registration of the multispectral image cube, allowing correction for patient motion during the thirty second data acquisition. To this end, we added a 92:8 beam splitter and a low-cost monochrome CCD camera in parallel with the commercial multispectral camera. The hardware developed for this application could be easily adapted to incorporate a second camera into the instrument for fluorescence guided surgery;this second camera could be used either for a second fluorescence image, in order to provide a simple autofluorescence correction, or for a pseudo-white light image of the surgical field. This year, we contributed to development of a third instrument, a small portable fluorescent camera for use on excised tissue in or near the surgical theater. The camera uses low-cost LED illumination, has user-friendly control via a USB connection to a laptop, and is otherwise entirely self-contained. It is intended for use in a hospital setting, permitting the evaluation of experimental fluorescent probes on excised tissue while avoiding the difficulties associated with removal of tissue specimens from the patient care setting as well as the extra time and environmental uncertainty associated with transport of specimens to a remote location. The instrument has been evaluated with an animal model;its first use in the clinical setting is expected within the next year. Also this year, we designed, assembled, and characterized a light source for use in evaluating the potential of several experimental fluorescent probes in photodynamic therapy. The light source is optimized for uniform illumination across a multiwell culture plate and ease of use.